Increase in concentration of intracellular free Ca2+ ions is a widely used signaling mechanism under various physiological conditions. Release of free Ca2+ ions inside the cell happens either through plasma membrane channel proteins that allow Ca2+ influx or by release from intracellular Ca2+ stores. Since Ca2+ release thus becomes the activity of a wide variety of proteins, methods for detection and measurement of intracellular free Ca2+ are used as assays for many proteins that are potential drug targets. These methods should be capable of sensing the transient rise in intracellular [Ca2+] and also of generating a detectable signal.
Most of the commonly used methods for Ca2+ sensing use molecules that exhibit changes in their fluorescence characteristics upon exposure to increase in [Ca2+]. These include small molecular weight macromolecules such as fura-2, indo-1, fluo-3, and Calcium-Green (U.S. Pat. Nos. 4,603,209 and 5,049,673) as well as proteins such as GFP (green fluorescent protein) family of proteins with a sensor peptide inserted within them (U.S. Pat. No. 6,469,154) or the cameleon molecules of tandem GFP constructs with an intervening Ca2+ sensing peptide sequence, that generate a FRET signal upon sensing free Ca2+ (U.S. Pat. No. 5,998,204). In all these methods, the fluorescence signal is also as short-lived as the Ca2+-transient, and hence signal acquisition in real-time is mandatory. Moreover this also imposes rapid liquid dispensing. These factors not only necessitates the use of more expensive equipments but also limits the choice of equipments that can be used in addition to imposing severe constraints for carrying out these assays at high throughput scale. U.S. Pat. No. 6,514,709 disclosed a method that prolongs the duration of the Ca2+-transients by incorporating an intracellular chelator that alters the kinetics of signal generation, so that the signal is prolonged. However in all these methods, measurements will have to be done on cells in the live condition.